restless, an Active Ac-like Transposon from the Fungus Tolypocladium inflatum: Structure, Expression, and Alternative RNA Splicing

Abstract

Elements of the hAT transposon family, such as the maize activator (Ac), have been discovered in a large number of eukaryotic species. This type of class II transposon, present in both plants and animals, has not been previously detected in any fungal organism. However, using a differential screening method to detect repetitive DNA, we have identified a hAT transposon in the hyphomycete Tolypocladium inflatum. The transposon, which we named restless, is 4,097 bp long, carries 20-bp inverted repeats and an 8-bp target site duplication, and encodes a long open reading frame which is interrupted by a single intronic sequence. The derived mRNA exhibits alternative splicing, resulting in the formation of two transcripts that may be translated into polypeptides of 157 or 803 amino acids. The predicted amino acid sequence of the larger polypeptide demonstrates significant homology with transposases from the hAT transposon family. A chromosomal analysis using pulsed-field gel electrophoresis showed that all seven chromosomal bands carry copies of the 4.1-kb transposon. This was confirmed in hybridization experiments with rare-cutting restriction endonucleases which indicate that about 15 copies are present in T. inflatum. The genomic distribution of restless and its transcriptional expression, alternative mRNA splicing, and genomic mobility all imply a potential role for this element in developing a transposon tagging system for use in filamentous fungi.Eukaryotic transposons can be divided into two major groups (18, 42). Class I transposable elements are able to transpose via an RNA intermediate, a process which employs a reverse transcriptase. Well-known members of this type include retrotransposons such as the copia element from Drosophila melanogaster (17) and the Ty elements from Saccharomyces cerevisiae (61).Class II elements, which transpose via DNA intermediates, demonstrate direct excision prior to their integration into target sequences. The best-known examples are the Ac-Ds elements of maize, discovered about half a century ago by Barbara McClintock (40,41).Both classes of transposons have previously been identified in filamentous fungi. In a few cases, such as the Neurospora crassa Tad element (37) and the Fusarium oxysporum Fot1 and impala elements (10, 39), this was achieved by the use of a transposon trap. Others were discovered by characterizing repeated DNA sequences, leading to the isolation of the MGR (24) and grasshopper (13) elements from Magnaporthe grisea, the Foret element from F. oxysporum (31), and the CfT-1 element from Cladosporium fulvum (43). The majority of these fungal transposons are class I transposable elements, while only a few belong to class II (Fot1 and impala). Those elements, whose prototypes were found in Caenorhabditis elegans or in D. melanogaster (7,28,46), were characterized as Tc1-mariner-like transposons (9).Here, we describe the isolation and characterization of a class II transposon from Tolypocladium inflatum ATCC 34921 (synonym Beauveria nivea [20,60]). This filamentou...

Hop, an Active Mutator-like Element in the Genome of the Fungus Fusarium oxysporum

“…The fact that Hop insertion does not determine a visible mutant phenotype suggests that this gene is not essential in F. oxysporum and/or is present in more than one copy. Additionally, a second Hop element is located 184 bp downstream of an ORF that we have partially sequenced, which encodes a putative protein strongly similar to transposases from fungal Ac-like transposons such as Tfo1 of F. oxysporum (Okuda et al 1998) and restless from Tolypocladium inflatum (Kempken and Kück 1996). This ORF is 99% identical to the putative product of a repetitive sequence (GenBank accession number X82490.1) reported to be responsible for the disruption of an F. oxysporum cytochrome P450 gene (Mouyna 1994).…”

“…Representatives of DNA transposons and retroelements have been characterized in these species, indicating that fungal transposons reflect the overall spectrum of eukaryotic transposable elements (for a review see Daboussi 1996;Daboussi 1997;Kempken and Kück 1998). Plant pathogenic fungi, such as Magnaporthe grisea, the main pathogen of rice, and Fusarium oxysporum, pathogenic on more than a hundred plant species, appeared to be very rich niches for TEs.…”

Heterologous transposition in Aspergillus nidulans

“…Others report the same lack of success with the niaD gene (J. R. Kinghorn, unpublished results). Recently, a number of putative or actually active transposons have been described in a number of fungi, such as Fusarium oxysporum (Daboussi et al, 1992;Daboussi and Langin, 1994;Langin et al, 1995), Magnaporthe grisea (Farman et al, 1996) and Tolypocladium inflatum (Kempken and Kuck, 1996), but also including Aspergillus niger (Glayser et al, 1995;Amutan et al, 1996). Close to the brlA gene of A. nidulans, there is an intact transposon, highly similar to the Fot1 element of F. oxysporum (Kupfer et al, 1997).…”

In situ analysis of apoptosis in Aspergillus nidulans with ethidium bromide and acridine orange

“…In contrast to other eukaryotes, this fungus has cytoplasm compartments with 2 to 10 nuclei that communicate via septal pores and form hyphae lined with a cell wall. The cell structure makes the concomitant analysis of several nuclei possible (Morris, 1975;Kempken and Kuck, 1996).…”